U.S. patent application number 11/918390 was filed with the patent office on 2009-11-19 for method for producing, and a substrate with, a surface with specific characteristics.
Invention is credited to Jas Pal S. Badyal, Wayne C. Schofield, Declan Oliver H. Teare.
Application Number | 20090286435 11/918390 |
Document ID | / |
Family ID | 34611112 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286435 |
Kind Code |
A1 |
Badyal; Jas Pal S. ; et
al. |
November 19, 2009 |
Method for Producing, and a Substrate with, a Surface with Specific
Characteristics
Abstract
The invention relates to a method of rendering the surface of a
substrate, or at least part of the substrate, to have increased
protein resistance. This is achieved by applying an n-substituted
glyconic derivative onto the surface of the substrate, or areas or
domains of the substrate surface, to allow the pattern resistance
to be changed in those areas where the material is applied. In one
embodiment the deposition is performed in or in conjunction with a
plasma.
Inventors: |
Badyal; Jas Pal S.; (County
Durham, GB) ; Teare; Declan Oliver H.; (Old Elvet,
GB) ; Schofield; Wayne C.; (Chester, GB) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
34611112 |
Appl. No.: |
11/918390 |
Filed: |
April 12, 2006 |
PCT Filed: |
April 12, 2006 |
PCT NO: |
PCT/GB2006/001329 |
371 Date: |
June 3, 2009 |
Current U.S.
Class: |
442/59 ;
204/192.15; 427/569; 428/422; 428/426; 428/457; 428/500; 428/532;
428/537.1; 428/537.5; 428/689; 428/704 |
Current CPC
Class: |
D06M 10/08 20130101;
Y10T 428/31989 20150401; Y10T 428/31993 20150401; D06M 15/263
20130101; Y10T 428/31678 20150401; D06M 14/18 20130101; D06M 13/224
20130101; Y10T 428/31971 20150401; Y10T 428/31544 20150401; D06M
15/285 20130101; Y10T 442/20 20150401; B05D 1/62 20130101; Y10T
428/31855 20150401; D06M 15/233 20130101; D06M 10/025 20130101;
D06M 13/342 20130101; A61L 33/064 20130101 |
Class at
Publication: |
442/59 ;
204/192.15; 427/569; 428/422; 428/426; 428/457; 428/500; 428/532;
428/537.1; 428/537.5; 428/689; 428/704 |
International
Class: |
B32B 9/04 20060101
B32B009/04; C23C 14/34 20060101 C23C014/34; B05D 1/00 20060101
B05D001/00; B32B 27/06 20060101 B32B027/06; B32B 17/06 20060101
B32B017/06; B32B 15/04 20060101 B32B015/04; B32B 29/00 20060101
B32B029/00; B32B 21/04 20060101 B32B021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2005 |
GB |
0507537.9 |
Claims
1. A method of coating a surface with a material, via one or more
application steps, to increase the protein resistance of at least
part of the surface, wherein said material is an n-substituted
glycine derivative.
2. A method according to claim 1 wherein the n-substituted glycine
derivative includes one or more unsaturated polymerizable
functional groups.
3. A method according to claim 2 wherein the group is any or any
combination of vinyl, styrene, acrylate, methacrylate, acrylamide,
and/or the like.
4. A method according to claim 1 wherein the n-substituted glycine
derivative includes an acrylamide group.
5. A method according to claim 1 wherein the n-substituted glycine
derivative is a methyl ester of glycine.
6. A method according to claim 1 wherein the n-substituted glycine
derivative is or includes n-acryloylsarcosine methyl ester.
7. A method according to claim 1 wherein the material is provided
in a patterned manner on the surface areas or domain with increased
pattern resistance in comparison to other areas of the substrate
surface.
8. A method according to claim 1 wherein the n-substituted glycine
derivative is any or any combination of n-methyl-n-2-propenyl
methyl ester, acryloylsarcosine methyl ester, N-methoxyethylglycine
oligomers, sarcosine-based monomers, and/or other n-substituted
glycine derivatives.
9. A method according to claim 1 wherein the n-substituted glycine
derivative is polymerized in combination with other polymerizable
monomers to form n-substituted glycine copolymers.
10. A method according to claim 9 wherein monomers for
copolymerization include any or any combination of vinyls,
styrenes, acrylates, acrylamides, and/or the like.
11. A method according to claim 1 wherein the method includes
pulsed plasmachemical deposition of the material.
12. A method according to claim 1 wherein the method includes
low-power continuous-wave plasma deposition of the material.
13. A method according to claim 11 wherein pulsed plasmachemical
deposition constitutes the generation of active sites at the
surface and in the electrical discharge during a duty cycle
on-period, followed by polymerization reaction pathways proceeding
during an extinction period.
14. A method according to claim 13 wherein the active sites are
predominantly radicals.
15. A method according to claim 13 wherein the duty cycle on-period
lasts between 1-100 microseconds and the extinction period lasts
between 1-20 milliseconds.
16. A method according to claim 1 wherein the application steps are
solventless and/or substrate independent.
17. A method according to claim 1 wherein the application steps
include any or any combination of grafting by pre-irradiation of a
surface with ionizing radiation or plasma, grafting by surface
polymerization from an initiator layer, by free radical
polymerization, atom transfer free radical polymerization,
iniferter polymerization, ionic polymerization, and/or
photopolymerization.
18. A method according to claim 1 wherein the application steps
include any or any combination of surface physisorption or
chemisorption of pre-formed n-substituted glycine derivative
oligomers or polymers onto a solid surface.
19. A method according to claim 11 or 12 wherein the plasma
operates at low, sub-atmospheric or atmospheric pressure.
20. A method according to claim 19 wherein the n-substituted
glycine derivative is introduced into the plasma as a vapour or an
atomised spray of liquid droplets.
21. A method according to claim 19 wherein the monomer is
introduced into the plasma deposition apparatus continuously, or in
a pulsed manner.
22. A method according to claim 1 wherein a substrate to which the
protein resistant coating is applied is located substantially
inside a pulsed plasma during coating deposition.
23. A method according to claim 1 wherein the substrate is located
outside of a pulsed plasma.
24. A method according to claim 1 wherein the n-substituted glycine
derivative is directly excited within a plasma discharge.
25. A method according to claim 1 wherein a remote plasma
deposition methods is used, wherein the material enters the
deposition apparatus substantially downstream of the pulsed
plasma.
26. A method according to claim 11 or 12 wherein the plasma
comprises the n-substituted glycine derivative alone, substantially
in the absence of other compounds.
27. A method according to claim 26 wherein plasmas consisting of
n-substituted glycine derivative alone are achieved by first
evacuating the reactor vessel as far as possible, purging the
reactor vessel with the n-substituted glycine derivative for a
period of time sufficient to ensure that the vessel is
substantially free of other gases.
28. A method according to claim 27 wherein the temperature in the
plasma chamber is sufficiently high to allow sufficient material
monomer in gaseous phase to enter the plasma chamber.
29. A method according to claims 1 or 12 wherein materials
additional to the n-substituted glycine derivative are present
within the plasma.
30. A method according to claim 29 wherein said additive materials
are inert and act as buffers without any of their atomic structure
being incorporated into the growing plasma polymer.
31. A method according to claim 30 wherein said additive materials
are noble gases.
32. A method according to claim 29 wherein the additive materials
include other monomers so that the resultant coating formed on the
substrate is a copolymer.
33. A method according to claim 32 wherein monomers for use within
the method of the invention include organic, inorganic,
organo-silicon and organo-metallic monomers.
34. A method according to claim 1 wherein the method is employed
for use in bio-micro electromechanical systems and for coating
other biomaterial surfaces where an immune response is not
desired.
35. A method according to claim 1 wherein said method includes the
step of applying the material using a pulsed plasmachemical
deposition technique.
36. A method according to claim 35 wherein the material is an
n-substituted glycine derivative.
37. A method according to claim 1 wherein the increased protein
resistant surface is only provided at selected surface areas or
domains on the substrate surface.
38. A method according to claim 37 wherein the selection is
achieved by plasma depositing the coating through a mask or
template to produce a substrate surface, with the domains or areas
covered with protein resistant material juxtaposed with areas of
the substrate that have no protein resistant material applied
thereto.
39. A method according to claim 37 wherein the steps include
depositing the material over the entire surface of the substrate
and then rendering selected areas of the surface incapable of
protein resistance.
40. A method according to claim 37 wherein the material restricted
to specific surface domains is an n-substituted glycine
derivative.
41. A method according to claim 37 wherein the areas provided with
the protein resistant areas or domains are provided in register
with visually apparent markings, on the substrate.
42. A method of applying a material to a substrate, via one or more
application steps, to increase the protein resistance of the
surface, wherein said material which is applied is a
poly(n-acryloylsarcosine methyl ester).
43. A method according to claim 37 wherein the method results in a
product wholly coated in a protein resistant polymer coating.
44. A substrate having an outer surface formed at least partially
of an n-substituted glycine derivative applied in accordance with
the method of claim 1.
45. A substrate according to claim 44 wherein the substrate has a
layer of n-substituted glycine derivative applied thereto to form
an outer surface thereof.
46. canceled.
47. A substrate according to claim 44 wherein the substrate
includes a protein resistant is formed of any or any combination of
woven or non-woven fibres, natural fibres, synthetic fibres, metal,
glass, ceramics, semiconductors, cellulosic materials, paper, wood,
or polymers such as polytetrafluoroethylene, polythene or
polystyrene.
48. (canceled)
49. (canceled)
Description
[0001] The invention to which this application relates is a method
of depositing a material onto a substrate to form a surface having
specific characteristics and particularly, although not necessarily
exclusively, the development of a surface layer which is
protein-resistant on at least portions thereon.
[0002] Although the following description refers almost exclusively
to use of an n-substituted glycine derivative to coat a substrate
surface, it will be appreciated by persons skilled in the art that
other substances can be utilised to increase the protein resistance
of a surface.
[0003] The forces which are understood to govern protein adsorption
onto a solid surface comprise hydrogen bonding, hydrophobic- and
electrostatic- interactions. Hydrogen bonds tend to form between
polar groups contained in the protein and which are present on the
surface. Hydrophobic forces arise due to the formation of a water
depletion zone at the interface between hydrophobic regions on a
protein molecule and a hydrophobic substrate, whilst electrostatic
interactions are associated with solvated charged groups on the
protein surface and the solid substrate. Once adsorbed onto a
surface, a protein may either stay in its natural conformation, or
denature (unfold), and such binding can be irreversible. For
instance, in the case of hydrophobic substrates, proteins usually
unfold in order to maximize interactions with the surface.
[0004] Since the late 1980s there has been a growing interest in
surfaces and coatings which resist this phenomena which is called
bio-adhesion (i.e. proteins, cells, and bacteria). The most widely
studied systems for minimising bio adhesion have been based upon
polyethylene oxide (PEO)/polyethylene glycol (PEG), phospholipids,
polysacchatides, and polyacrylamides. As a general rule, surfaces
which minimize protein adsorption prove to be resistant towards
cell attachment and tissue culture growth.
[0005] PEO/PEG surfaces are considered to be the benchmark
performers for minimizing protein adhesion. The non-fouling
character of PEO surfaces is attributed to the very high levels of
polymer chain hydration as well as the conformational flexibility
of the polymer. A number of methods exist for making PEO surfaces;
these include: gold-, silicon-, silica-, and diamond-based
self-assembled monolayers (SAMs), physisorption, chemisorption,
surface initiated polymerization, plasma initiated grafting,
covalent grafting onto a plasma polymer, and plasma polymerization.
A PEO-mimicking thiol-functionalized polyester with ether side
chains SAM on gold has also been shown to display minimal protein
adsorption characteristics.
[0006] However, many of these systems have intrinsic disadvantages:
PEO suffers from a susceptibility towards oxidative degradation and
chain cleavage in aqueous environments (PEO coatings can degrade
and lose their bio-inertness after several days of immersion in
buffer). Alkanethiol-gold-based SAMs (which comprise the majority
of systems studied so far), such as alkanethiol-terminated gold
SAMs of sarcosine based polypeptides, have been shown to exhibit
good protein-resistant properties, but SAM systems are known to
suffer from being substrate-specific and tend to be unstable as a
consequence of their thiolate groups (Au-SR) being susceptible
towards oxidation and desorption from the gold surface, leading to
a complete loss of the protein-resistant properties.
[0007] Phospholipids are another extensively studied class of
molecules capable of rendering surfaces protein-resistant. These
biomimetic surfaces resemble the outer lipid membrane surface of
erythrocytes, and as such they are non-thrombogenic. In particular,
phosphorylcholine (the head group of lecithin) based surfaces have
been shown to improve biocompatibility. The protein resistance
behaviour of phosphorylcholine surfaces can be attributed to the
very high levels of hydration of the zwittetionic headgroup; these
positive and negative charges render the surface neutral over a
large pH range. This hydration layer ensures that proteins which
come into contact with the surface do so reversibly and without
deformation.
[0008] Many of the methods employed to produce phosphorylcholine
surfaces rely on the ability of these amphiphilic molecules to
self-assemble as planar supported lipid bilayers, (or multilayers).
This has been achieved by spin coating, Langmuit-Blodgett
deposition, and liposome adsorption. However, a major drawback
encountered with most of these systems is that they are reliant
upon weak van der Waals interactions (the phosphorylcholine
molecules themselves being only weakly associated with each other
and to the underlying substrate, thereby compromising the overall
bulk physical properties of the material). Attempts aimed at
improving the binding of phosphorylcholine-based films have
included grafting to plasma irradiated surfaces, forming SAMs of
phosphorylcholine terminated alkanethiols onto gold, cross-linking
the phosphorylcholine chains via diene groups in the alkyl chains,
and copolymerizing phosphorylcholine-methacrylates with other
monomers.
[0009] Saccharide groups are also known for their protein-resistant
behaviour. In a similar manner to PEO/PEG and phospholipids, these
hydrophilic surfaces are highly hydrated and thus render the
substrate protein-resistant. For instance, dextran is reported to
be protein-resistant, and limits the adhesion and spreading of
cells, although absolute protein rejection is not observed.
Alkanethiol-based saccharide SAMs which have displayed protein
resistance comparable to PEO, include methylated sorbitol and
mannitol. The latter is capable of sustaining protein-resistant
behaviour for much longer periods of time compared to PEO based
SAMs (thus overcoming one of the principal disadvantages of
PEO).
[0010] Polyacrylamides are another category of protein-resistant
surface. In particular, the thermoresponsive polymer
poly(n-isopropylacrylamide) behaves as a protein-resistant material
below its lower critical solution temperature (LCST), and switches
to being protein-adsorbent above its LCST. A main disadvantage in
this case is that it absorbs protein at body temperature.
[0011] Other surfaces that have been found to exhibit protein
resistance include tripropylsulphoxide terminated alkanethiol SAMs
and elastin-like polypeptide coatings. Lately, there has been an
interest in various alternative protein-resistant alkanethiol-gold
SAMs based on kosmotropes (molecules that exclude themselves from
the protein-water interface) such as polyols, betaine, taurine,
trimethylamine-N-oxide, dimethyl acetamide, dimethyl sulphoxide,
and hexamethylphosphoramide.
[0012] Finally, there is an alternative strategy where adsorption
of serum albumin onto a surface can lead to the suppression of
protein adsorption. However, this method is not particularly
robust, and adsorbed proteins are vulnerable to eventual
displacement by more surface active proteins due to the Vroman
effect.
[0013] The aim of the present invention is to provide a method of
producing a protein-resistant surface which overcomes the above
issues.
[0014] In a first aspect of the invention, there is provided a
method of coating a surface with a material, via one or more
application steps, to increase the protein resistance of at least
part of the surface, wherein said material is an n-substituted
glycine derivative.
[0015] In one embodiment the n-substituted glycine derivative
includes one or more unsaturated polymerizable functional groups.
Typically the group is any or any combination of vinyl, styrene,
acrylate, methacrylate, acrylamide, and/or the like. In a preferred
embodiment the n-substituted glycine derivative includes an
acrylamide group.
[0016] In a preferred embodiment the n-substituted glycine
derivative is a methyl ester of glycine.
[0017] In one embodiment the n-substituted glycine derivative is or
includes n-acryloylsarcosine methyl ester.
[0018] In accordance with the invention the instability of the SAM
system is thus overcome by adopting an acrylamide form of sarcosine
for polymerization to produce a protein-resistant film.
[0019] In an alternative embodiment the n-substituted glycine
derivative is any or any combination of n-methyl-n-2-propenyl
methyl ester, acryloylsarcosine methyl ester, N-methoxyethylglycine
oligomers, sarcosine-based monomers, and/or other n-substituted
glycine derivatives.
[0020] In an alternative embodiment the n-substituted glycine
derivative is polymerized in combination with other polymerizable
monomers to form n-substituted glycine copolymers. Typically,
monomers for co-polymerization include any or any combination of
vinyls, styrenes, acrylates, acrylamides, and/or the like.
[0021] In one embodiment the application method includes pulsed
plasmachemical deposition. In another embodiment the application
includes low-power continuous-wave plasma deposition.
[0022] Typically pulsed plasmachemical deposition constitutes the
generation of active sites at the surface and in the electrical
discharge during a duty cycle on-period, followed by conventional
polymerization reaction pathways proceeding during an extinction
period. Typically the active sites are predominantly radicals.
[0023] Typically the duty cycle on-period lasts 1-100 microseconds.
Typically the extinction period lasts 1-20 milliseconds.
[0024] Typically the application steps are solventless and/or
substrate independent.
[0025] In an alternative embodiment the application steps include
any or any combination of grafting by pre-irradiation of a surface
with ionizing radiation or plasma, grafting by surface
polymerization from an initiator layer, by free radical
polymerization, atom transfer free radical polymerization,
iniferter polymerization, ionic polymerization, and/or
photopolymerization.
[0026] In an alternative embodiment the application method includes
any or any combination of the steps of surface physisorption or
chemisorption of pre-formed n-substituted glycine derivative
oligomers or polymers onto a solid surface.
[0027] Typically the plasma operates at low, sub-atmospheric or
atmospheric pressure. In one embodiment the n-substituted glycine
derivative is introduced into the plasma as a vapour or an atomised
spray of liquid droplets. In one embodiment the monomer is
introduced into the pulsed plasma deposition apparatus
continuously, or in a pulsed manner by way of, for example, a gas
pulsing valve.
[0028] In one embodiment the substrate to which the protein
resistant coating is applied is located substantially inside the
pulsed plasma during coating deposition. Alternatively the
substrate may be located outside of the pulsed plasma, thus
avoiding excessive damage to the substrate or growing coating.
[0029] Typically the n-substituted glycine derivative is directly
excited within the plasma discharge. Alternatively, remote plasma
deposition methods may be used, wherein the monomer enters the
deposition apparatus substantially downstream of the pulsed plasma,
thus reducing the potentially harmful effects of bombardment by
short-lived, high energy species such as ions.
[0030] In one embodiment the plasma comprises the n-substituted
glycine derivative alone, substantially in the absence of other
compounds. Plasmas consisting of n-substituted glycine derivative
alone may be achieved by first evacuating the reactor vessel as far
as possible, and then purging the reactor vessel with the
n-substituted glycine derivative for a period sufficient to ensure
that the vessel is substantially free of other gases. Typically the
temperature in the plasma chamber is sufficiently high to allow
sufficient monomer in gaseous phase to enter the plasma chamber.
This will depend upon the monomer and conveniently ambient
temperature will be employed. However, elevated temperatures for
example from 25 to 250.degree. C. may be required in some
cases.
[0031] In alternative embodiments of the invention, materials
additional to the n-substituted glycine derivative are present
within the plasma deposition apparatus. The additional materials
may be introduced into the coating deposition apparatus
continuously or in a pulsed manner by way of, for example, a gas
pulsing valve. Typically said additive materials are inert and act
as buffers without any of their atomic structure being incorporated
into the growing plasma polymer. Typically said additive materials
are noble gases. A buffer of this type may be necessary to maintain
a required process pressure and/or sustain the plasma discharge.
For example, the operation of atmospheric pressure glow discharge
(APGD) plasmas often requires large quantities of helium. This
helium diluent maintains the plasma by means of a Penning
Ionisation mechanism without becoming incorporated within the
deposited coating.
[0032] In alternative embodiments of the invention, the additive
materials may be other monomers such that the resultant coatings
comprise copolymers. Suitable monomers for use within the method of
the invention include organic, inorganic, organo-silicon and
organo-metallic monomers.
[0033] In one embodiment the method also improves resistance
towards cell adhesion, bacteria adhesion, and/or enzyme
degradation. The method could be employed for use in bio-micro
electromechanical systems and for coating other biomaterial
surfaces where an immune response is not desired.
[0034] In a further aspect of the invention, there is provided a
method of applying a material to a substrate to increase the
protein resistance of at least part of the substrate surface formed
by the applied material, wherein said method includes the step of
applying the material using a pulsed plasmachemical deposition
technique.
[0035] In one embodiment the material is an n-substituted glycine
derivative.
[0036] In a further aspect of the invention, there is provided a
method of applying a material to a substrate, via one or more
application steps, to increase the protein resistance of the
surface, wherein said material which is applied is a
poly(n-acryloylsarcosine methyl ester).
[0037] Typically the method results in a product wholly coated in a
protein resistant polymer coating.
[0038] Alternatively the protein resistant polymer coating is only
applied to selected surface areas or domains. Typically the
restriction of the protein resistant polymer coating to specific
surface domains is achieved by limiting the means of coating
production of the method to said specific surface domains. In one
embodiment the restriction is achieved by plasma depositing the
coating through a mask or template. This produces a surface
exhibiting regions covered with protein resistant polymer
juxtaposed with regions that exhibit no protein resistant
polymer.
[0039] An alternative means of restricting the protein resistant
behaviour of the polymer coating to specific surface domains
comprises: depositing the protein resistant polymer over the entire
surface of the sample or article, before rendering selected areas
of it incapable of protein resistance. The spatially selective
removal/damage of the protein resistant polymer material which has
been applied may be achieved using electron beam etching and/or
exposure to ultraviolet irradiation through a mask. The pattern of
non-transmitting material possessed by the mask is hence
transferred to areas of protein resistance.
[0040] In one embodiment the material restricted to specific
surface domains is an n-substituted glycine derivative.
[0041] In a further aspect of the invention there is provided a
substrate having an outer surface formed at least partially of an
n-substituted glycine derivative.
[0042] In one embodiment the substrate has a layer of n-substituted
glycine derivative applied thereto to form an outer surface
thereof.
[0043] In one embodiment the substrate includes a protein resistant
coating obtained by a process as described above, said substrate
including any solid, particulate, or porous substrate or finished
article, typically consisting of any or any combination of
materials such as, but not limited to, woven or non-woven fibres,
natural fibres, synthetic fibres, metal, glass, ceramics,
semiconductors, cellulosic materials, paper, wood, or polymers such
as polytetrafluoroethylene, polythene or polystyrene. In a
particular embodiment, the surface comprises a polymeric support
material, capable of use in biochemical analysis or in vitro.
[0044] In a further aspect of the invention there is provided a
method of forming a protein resistant outer surface of a substrate,
said method including the step of applying an n-substituted glycine
derivative onto at least part of a surface of said substrate using
a pulsed plasmachemical deposition procedure.
[0045] A specific embodiment of the invention is now described
hereinbelow wherein;
[0046] FIG. 1 illustrates the polymerization of n-acryloylsarcosine
methyl ester to form a surface coating in accordance with one
embodiment of the invention.
[0047] FIG. 2 illustrates XPS C(1s) envelopes of
poly(n-acryloylsarcosine methyl ester): (a) 30 W continuous wave
plasma deposited; (b) pulsed plasma deposited (P.sub.p=30 W,
t.sub.ON=20 .mu.s, t.sub.off=5 ms); and (c) theoretically
predicted.
[0048] FIG. 3 illustrates FTIR spectra of: (a) 30 W continuous wave
plasma polymerized n-acryloylsarcosine methyl ester; (b) pulsed
plasma (P.sub.p=30 W, t.sub.ON=20 .mu.s, t.sub.off=5 ms)
polymerized n-acryloylsarcosine methyl ester; and (c)
n-acryloylsarcosine methyl ester monomer.
[0049] FIG. 4 illustrates SPR of protein adsorption onto plasma
polymerized n-acryloylsarcosine methyl ester films: (a) fibrinogen;
(b) lysozyme; and (c) Alexa-fluor 633 IgG. (P.sub.p=30 W (CW and
pulsed), t.sub.on=20 .mu.s, and t.sub.off=5 ms).
[0050] FIG. 5 illustrates fluorescence micrographs of pulsed plasma
deposited poly(n-acryloylsarcosine methyl ester) arrays following
immersion in Alexa-fluor 633 IgG/PBS solution: (a) embossed pattern
(negative image); and (b) UV exposed pattern (positive image).
(P.sub.p=30 W, t.sub.ON=20 .mu.s, and t.sub.off=5 ms).
[0051] FIG. 6 illustrates fluorescent microscope images of a
micro-spotted array produced on NASME pulsed plasma polymer layer:
(a) Protein Probe II; (b) Protein Probe IV;
[0052] FIG. 7 illustrates fluorescent microscope images of a
micro-spotted array produced on NASME pulsed plasma polymer layer:
(a) Protein Probe I subsequently exposed to Probe II; (b) Protein
Probe III subsequently exposed to probe IV; and
[0053] FIG. 8 illustrates fluorescent microscope image of a
micro-spotted array produced on NASME pulsed plasma polymer layer:
(a) alternating microarray pattern of Protein Probe I and III
subsequently exposed to probe II; (b) alternating microarray
pattern of Protein Probe I and III subsequently exposed to protein
Iv.
[0054] With reference to FIG. 1, there is illustrated a method in
accordance with one embodiment of the invention in which a pulsed
plasma polymerization 6 of n-acryloylsarcosine methyl ester (NASME)
2 is performed to form a polymerized surface coating 4 on a
substrate.
[0055] Specific method parameters and experimental details are set
out below;
[0056] N-acryloylsarcosine methyl ester (97%, Lancaster) monomer 2
was loaded into a sealable glass tube and further purified using
multiple freeze-pump-thaw cycles. Plasma polymerization 6 was
carried out in a cylindrical glass reactor (4.5 cm diameter, 460
cm.sup.3 volume, 2.times.10.sup.-3 mbar base pressure, and
1.4.times.10.sup.-9 mol s.sup.-1 leak rate), surrounded by a copper
coil (4 mm diameter, 10 turns, located 15 cm away from the
precursor inlet) and connected to a 13.56 MHz radio frequency (RF)
power supply via an L-C matching network. The reactor was located
inside a temperature controlled oven and a Faraday cage. A 30 L
min.sup.-1 rotary pump attached to a liquid nitrogen cold trap was
used to evacuate the plasma chamber. System pressure was monitored
with a Pitani gauge. All fittings were grease free. During pulsed
plasma deposition 6, the RF power source was triggered by a signal
generator and the pulse shape monitored with an oscilloscope. Prior
to each experiment, the apparatus was scrubbed with detergent,
rinsed with propan-2-ol, and oven dried. Further cleaning entailed
running a continuous wave air plasma at 0.2 mbar pressure and 40 W
power for 20 min. Next, silicon wafers, gold chips, or cut
polystyrene squares (15 mm.times.15 mm) were inserted into the
reactor and the system pumped down to base pressure. A continuous
flow of n-acryloylsarcosine methyl ester vapour was introduced into
the chamber at a pressure of 0.1 mbar and 40.degree. C. temperature
for 5 min prior to plasma ignition. The optimum pulsed plasma duty
cycle corresponded to 30 W peak power (P.sub.p) continuous wave
bursts lasting 20 .mu.s (t.sub.on) followed by an off-period
(t.sub.off) set to 5 ms. Once deposition was completed, the RF
power was switched off, and the monomer allowed to continue to
purge through the system for a further 5 min prior to evacuating to
base pressure and venting to atmosphere.
[0057] A spectrophotometer (nkd-6000, Aquila Instruments Ltd.) was
used to measure plasma polymer film 4 thickness and deposition
rate. The obtained transmittance-reflectance curves (350-1000 nm
wavelength range) were fitted to a Cauchy model using a modified
Levenberg-Marquardt algorithm.
[0058] Contact angle analysis of the plasma deposited
n-acryloylsarcosine methyl ester films was carried out with a video
capture system (ASE Products, model VCA2500XE) using 2.0 .mu.l
droplets of deionized water.
[0059] X-ray photoelectron spectroscopy (XPS) was undertaken using
an electron spectrometer (VG ESCALAB MK II) equipped with a
non-monochromated Mg K.alpha. X-ray source (1253.6 eV) and a
concentric hemispherical analyzer. Photo-emitted electrons were
collected at a take-off angle of 30.degree. from the substrate
normal, with electron detection in the constant analyzer energy
mode (CAE, pass energy=20 eV). The XPS spectra were charge
referenced to the C(1s) peak at 285.0 eV and fitted with a linear
background and equal full-width-at-half-maximum (FWHM) Gaussian
components using Marquardt minimization computer software.
Instrument sensitivity (multiplication) factors derived from
chemical standards were taken as being C(1s): O(1s): N(1s)=1.00:
0.36: 0.57.
[0060] The stoichiometry of the plasma deposited
poly(n-acryloylsarcosine methyl ester) films was determined by XPS,
as indicated in Table 1.
TABLE-US-00001 TABLE 1 XPS elemental analysis and water contact
angles for plasma deposited poly(n-acryloylsarcosine methyl ester).
XPS Contact Conditions % C % N % O angle/.degree. Continuous Wave
72 .+-. 0.6 9 .+-. 0.1 19 .+-. 0.5 41 .+-. 1.7 Pulsed 66 .+-. 0.5 9
.+-. 0.1 25 .+-. 0.6 44 .+-. 0.9 Theoretical 64 9 27 N/A (P.sub.p =
30 W (CW and pulsed), t.sub.on = 20 .mu.s, and t.sub.off = 5
ms).
With reference to FIGS. 1-2, pulsing the plasma at a duty cycle of
P.sub.p=30 W, t.sub.on=20 .mu.s, and t.sub.off=5 ms produced films
most resembling the theoretically predicted structure. The absence
of any Si(2p) XPS signal from the underlying silicon or gold
substrates verified pinhole-free film thicknesses exceeding the XPS
sampling depth (2-5 nm).sup.i.
[0061] The XPS C(1s) envelope can be fitted to 6 carbon
environments: hydrocarbon (C.sub.xH.sub.y=285.0 eV, .alpha.),
carbon adjacent to a carbonyl group (C--C.dbd.O=285.7 eV, .beta.),
carbon attached to nitrogen (C--N=286.2 eV, .chi.), carbon attached
to oxygen (C--O=286.6 eV, .delta.), carbon attached to nitrogen and
oxygen (N--C.dbd.O=288 eV, .epsilon.), and carbon attached to two
oxygens (O--C.dbd.O=289.3 eV, .phi.). 30 W continuous wave plasma
deposited n-acryloylsarcosine methyl ester films display a high
level of disruption of monomer structure as evident from the
elemental composition and the distribution of C(1s) components, as
shown in Table 1 and FIG. 2. Much better structural retention was
achieved during low duty cycle pulsed plasma deposition
[0062] With reference to FIG. 3, structural retention for the
pulsed plasma deposited poly(n-acryloylsarcosine methyl ester)
films was also authenticated by infrared spectroscopy. Surface
infrared spectroscopy of plasma polymer coated gold slides was
performed using an FTIR spectrometer (Perkin Elmer, model Spectrum
One) equipped with a liquid nitrogen cooled MCT detector operating
at 4 cm.sup.-1 resolution over the 700-4000 cm.sup.-1 range. A
reflection absorption accessory (RAIRS, Specac) and a I(RS-5
p-polarizer were fitted to the instrument, with the reflection
angle set to 80.degree..
[0063] Characteristic absorption bands include 1749 cm.sup.-1
(ester carbonyl), 1653 cm.sup.-1 (amide I), and 1212 cm.sup.-1
(ester C--O). The carbon-carbon double bond absorption at 1615
cm.sup.-1 (dashed line marked with *) associated with the monomer
is not present in any of the plasma deposited films, thus
indicating complete polymerization of the precursor. 30 W
continuous wave plasma deposition conditions yielded broad infrared
absorption features, which can be taken as being symptomatic of a
loss of monomer structural integrity. The low duty cycle pulsed
plasma deposited film (P.sub.p=30 W, t.sub.on=20 .mu.s, and
t.sub.off=5 ms) displays much better resolved absorption bands
matching those seen for the monomer (apart from the polymerizable
carbon-carbon double bond stretch at 1615 cm.sup.-1), thereby
confirming a high degree of structural retention.
[0064] Referring to FIG. 4, SPR analysis of fibrinogen and lysozyme
adsorption onto 25 nm thick pulsed plasma deposited
poly(n-acryloylsarcosine methyl ester) films (P.sub.p=30 W,
t.sub.on=20 .mu.s, and t.sub.off=5 ms) displayed excellent
resistance towards protein adsorption.
[0065] Surface plasmon resonance (SPR) protein adsorption studies
entailed plasma deposition of 25 nm thick poly(n-acryloylsarcosine
methyl ester) films onto a gold sensor chip (Biacore) and
monitoring protein adsorption using a biosensor SPR system (Biacore
1000 upgrade). Fibrinogen (from bovine plasma, Sigma) and lysozyme
(egg white, Sigma) proteins were screened. Fibrinogen is a large
protein which interacts with platelets during blood clotting; it is
a good example of a "sticky protein". Lysozyme is smaller and
positively charged under the experimental conditions used, and is
often employed as a model protein to study electrostatic
adsorption. The experimental protocol for measuring protein
adsorption entailed firstly ensuring a clean surface by flowing a
40 mM in phosphate buffered saline solution of sodium dodecyl
sulphate (+99%, Sigma) over the surface for 3 min followed by
flushing with phosphate buffered saline for 10 min. Next, the
protein solution (1 mg ml.sup.-1 in phosphate buffered saline, pH
7.4) was passed over the surface for 30 min. Finally, phosphate
buffered saline was flushed through the system for 10 min in order
to dislodge any loosely-bound proteins. The flow rate for all SPR
experiments was set at 10 .mu.l min.sup.-1. In all cases, the
buffer was de-gassed and filtered using a 200 nm cellulose nitrate
filter (Whatman) prior to use.
[0066] The films remained protein resistant at body temperature
(i.e. 36.degree. C.). In contrast, continuous wave plasma
deposition conditions gave rise to approximately two orders of
magnitude greater protein adsorption. UV irradiation of the pulsed
plasma deposited poly(n-acryloylsarcosine methyl ester) for 40 min
was sufficient to change the previously protein-resistant films to
being as protein-receptive as the continuous wave film.
[0067] With reference to FIG. 5, SPR analysis of the adsorption of
the fluorescent marker Alexa-fluor 633 IgG protein independently
confirmed the good protein-resistance properties of the pulsed
plasma deposited poly(n-acryloylsarcosine methyl ester) films
illustrated in FIG. 4.
[0068] Alexa-fluor 633 goat anti-mouse immunoglobulin (IgG, 2 mg
ml.sup.-1 in phosphate buffered saline, Molecular Probes) further
diluted to a concentration of 250 .mu.g ml.sup.-1 in phosphate
buffered saline was employed as a fluorescent marker for mapping
patterned arrays of pulsed plasma deposited
poly(n-acryloylsarcosine methyl ester) films by fluorescent
microscopy. Negative image protein arrays were created by embossing
nickel grids (2000 mesh, 7.5 .mu.m holes with 5 .mu.m bars, Agar
Scientific) into polystyrene plates using a weight of 4 tons for 10
s, followed by pulsed plasma deposition of poly(n-acryloylsarcosine
methyl ester). The nickel grid was then lifted off from the
polystyrene substrate to leave behind a well-defined array of
plasma polymer. Positive image protein arrays were created by
pulsed plasma depositing poly(n-acryloylsarcosine methyl ester)
films onto a blank polystyrene chip and then irradiating through a
nickel mask (2000 mesh, 7.5 .mu.m holes with 5 .mu.m bars, Agar
Scientific) using a wide band HgXe UV source arc-lamp (Oriel model
6136) operating at a power of 0.3 W cm.sup.-1 for 40 min. All
patterned chips were subsequently immersed into a 250 .mu.g
ml.sup.-1 solution of Alexa-fluor 633 IgG in phosphate buffered
saline for 60 min. This was followed by successive rinses in
phosphate buffered saline, 50% phosphate buffered saline diluted
with deionized water, and finally twice with deionized water prior
to fluorescent microscopy analysis. A Raman microscope system
(LABRAM, Jobin Yvon) was used to collect a two-dimensional
fluorescent map of the Alexa-fluor 633 IgG protein patterned
surfaces. This entailed focusing an unattenuated 633 nm He--Ne
laser beam (20 mW) onto the sample using a microscope objective
(.times.50) and the corresponding fluorescence signal collected
through the same objective via a back-scattering configuration in
combination with a cooled CCD detector. The diffraction grating was
set at 300 groves mm.sup.-1 with the laser filter at 100%
transmission. The sample was mounted onto a computerized X-Y
translational mapping stage and the surface rastered (50
.mu.m.times.50 .mu.m) using a 1 .mu.m step size.
[0069] Fluorescence microscopy of the embossed array of pulsed
plasma deposited poly(n-acryloylsarcosine methyl ester) following
the adsorption of Alexa-fluor 633 IgG protein (negative image)
showed clear contrast in signal intensity between the regions of
plasma polymer (dark squares) and the uncoated polystyrene (bright
grid). Whereas fluorescence microscopy of the UV patterned pulsed
plasma deposited poly(n-acryloylsarcosine methyl ester) array after
exposure to Alexa-fluor 633 IgG protein (positive image) only
displayed signal intensity corresponding to UV exposure (bright
squares) and not from the unexposed areas (dark grid).
[0070] In summary, proteins in aqueous solution present hydrophilic
groups at the protein-water interface, and any of these interfacial
functionalities which are charged will attract an electric double
layer of ions from the surrounding solution to screen the charge.
Along with these counterions a sheath of structured water molecules
surrounding the protein will exist. Likewise, a hydrophilic
molecule such as protein-resistant PEO, phosphorylcholine, or
zwitterionic sulphobetaine will possess a similar surrounding
sheath of ordered water molecules, as verified by Raman
spectroscopy. These protein-resistant moieties are understood to
disrupt the ordering of water molecules in the domain local to a
protein significantly less than non-protein-resistant substrates
(such as poly(hydroxyethyl methacrylate), sodium
poly(ethylenesulfonate), and poly-L-lysine). Thus in the former
case, any long-range attractive forces between the protein and the
surface are insufficient to overcome the steric repulsion
encountered when the structured water interface around the protein
and the surface try to overlap, and hence the surface is rendered
protein-resistant, i.e. it has an excluded volume. Other factors
such as the packing, alignment and flexibility of the surface
molecules may also be taken into account to effect the protein
resistance.
[0071] Pulsed plasma deposited poly(n-acryloylsarcosine methyl
ester) is hydrophilic with a contact angle of 44.+-.1.7.degree., as
indicated by Table 1 above. This hydrophilicity stems from the
terminal ester group and the polymer backbone amide linkages.
Furthermore, the polymer does not contain any groups with
hydrogen-bond donating capacity, i.e. it obeys the set of four
molecular criteria postulated by Whitesides for protein resistance;
these being the presence of (i) polar functional groups, (ii)
hydrogen bond accepting groups, (iii) the absence of hydrogen bond
donating groups, and (iv) no net charge. Therefore it seems highly
probable that the hydrated surface of poly(n-acryloylsarcosine
methyl ester) films forms an exclusive volume to proteins that
renders it protein-resistant.
[0072] Further tests (not shown) have shown that the long-term
protein resistance of the poly(n-acryloylsarcosine) surface does
not deteriorate over several months. The plasma polymer is not
susceptible towards oxidative degradation when placed in either air
or phosphate buffered saline solution, thus avoiding the
disadvantage associated with PEO films. Furthermore, it benefits in
that--unlike poly(n-isopropylacrylamide)--this polymer remains
protein-resistant at body temperature (36.degree. C.). In the case
of pulsed plasmachemical deposition, the coating is also
substrate-independent, giving applicability to diverse uses such as
protein-resistant biomaterials and proteomics chips. The coating
may be applied to gold, glass, silicon, polystyrene microspheres,
and polymer non-wovens. Furthermore, by the application of the
coating material onto the substrate through a mask or template so
specific domains or areas of the substrate can be defined to have
the protein resistant characteristics thus allowing the generation
of specific areas in which samples can be applied and held in these
specific areas. In one embodiment these specified areas can be
further defines by visually apparent grid patterns or other
markings which are present on the substrate, perhaps as a result of
printing with the markings in register with the said areas of
domain.
[0073] In a further example of the invention there is provided
results which show the use of the invention for Protein adsorption
onto locally activated NASME surfaces for better definition Protein
Arrays
TABLE-US-00002 TABLE 2 Proteins and fluorophores used in this
study. Fluorophore Protein attached to Label Protein protein Probe
I IgG from equine serum, reagent N/A grade >= 95%, lypophilized,
essentially salt-free (Molecular Probes Inc) Probe II Protein A
from staphylococcus FITC aureus (Sigma-Aldrich Ltd) Probe III
Protein G from streptococcus sp, N/A lypophilized from a Tris-HCl
buffer (Sigma-Aldrich Ltd) Probe IV Goat antimouse IgG (H + L)
Alexa fluor 633 2 mg/L in 0.1M NaP, 0.1M NaCl pH 7.5 5 mM azide
[0074] In experimentation in this case protein immobilization to
pulsed plasma polymerised NASME surfaces entailed immersing a
protein Probe into a buffer pH=7.5 (consisting of 200 nM sodium
hydroxide, 2% Ficoll 400, 2% polyvinylpyrollidone (PVP), 0.5%
sodium dodecyl sulphate (SDS) and sodium chloride/sodium citrate (3
M NaCl, 0.3 M Na Citrate--2H.sub.2O, Aldrich)) to a final
concentration of 20 .mu.g/mL. The buffered solution was placed onto
freshly prepared pulsed plasma poly(NASME) surfaces using a robotic
microarrayer (Genetix Inc) equipped with micro-machined pins that
consistently delivered samples of .about.1 nL (20 .mu.g/L buffered
protein solution) onto the pulsed plasma poly(NASME) coated glass
slides (18.times.18.times.0.17 mm, BDH) at designated locations.
Typical circular spots with diameter ranging from 250-300 .mu.m and
with a minimum print pitch of 900 .mu.m could be routinely
obtained. After this "spotting" process, the protein immobilised
slides were kept in a humidity chamber (64% relative humidity) for
72 hours at 37.degree. C. Finally, the slides were removed and
washed with buffer solution and copious amounts of water for a
further 72 hours. Probe I, II, III and IV (Table 2) were used in
the spotting process. A further microarray pattern was developed
consisting of spots of alternating probe I and III.
[0075] Protein microarrays of Probe I and III were subsequently
exposed to a single complementary protein solution of probe II and
probe IV, respectively, dissolved into a phosphate buffered saline
solution (pH=7.0 Sigma-Aldrich Ltd) to a final concentration of 20
.mu.g/mL. Two small strips of adhesive tape were affixed along the
rim of Probe I and III patterned slides and then covered with a
cleaned microscope slide cover glass. The cavity formed between the
chip and the cover glass was filled by slowly loading 10 .mu.L of
the complementary solution by capillary force. The slide was
incubated in a humidified chamber was immersed in at 37.degree. C.
for 24 hours. After the cover glass was removed, the chip was
washed three times with buffer solution and then with copious
amounts of water (48 hours) and blow dried with nitrogen gas.
[0076] Fluorescent microscopy mapping was performed using an
Olympus IX-70 microscope driven by the SoftWorx package system
(DeltaVision RT, Applied Precision). Image data was collected using
excitation wavelengths at 525 nm and 633 nm corresponding to the
absorption maxima of the dye molecules, FITC and alexa fluor 633
respectively. Imaging was performed using .times.10 objective using
Openlab software (Improvision). Finally, Images were deconvolved
using SoftWorx and quick projections saved as Adobe Photoshop
images.
[0077] In the case of immobilisation of proteins Probe II and IV,
fluorescent images of an array of spots with an average diameter of
250 microns and a print pitch of 900 microns is in accord with the
spotting parameters used and images are shown in FIGS. 6(a) and
(b). In the case of complementary arrays, a similar pattern was
obtained on the surface; however, the spot size had increased to
300 microns in diameter and the spots have an inhomogeneous outer
edge density signal as shown in FIGS. 7(a) and (b). Upon exposure
of the alternating microarray pattern of probe I and III to probe
II only binding to probe I was observed as shown in FIG. 8(a).
Additionally, upon exposure of the alternating microarray to probe
IV, only binding to probe III was observed, FIG. 8(b).
[0078] The benefit of this particular, localised, NASME activation
method is that the region surrounding each immobilised protein spot
remains protein-resistant, thereby offering superior definition
compared to previous methods.
[0079] It will be appreciated by persons skilled in the art that
the present invention also includes further additional
modifications made to the method which does not effect the overall
functioning of the method.
* * * * *